A protein fraction which lacks DNA-binding activity itself, but confers enhanced protein-DNA complex formation to E. coli core RNA polymerase, was obtained from mustard chloroplasts by heparin Sepharose chromatography. Gel retardation and competition assays as well as DNase I footprinting experiments with a chloroplast DNA fragment containing the psbA promoter indicate that this reflects sequence-specific binding. Transcription of the psbA template by E. coli core enzyme in the presence of the chloroplast fraction results in enhanced formation of transcripts of the size expected for correct initiation at the in vivo start site. We conclude that the chloroplast fraction reveals sigma-like activity with E. coli RNA polymerase and thus might contain factor(s) of equivalent function in chloroplast transcription.